zurichopenrepositoryand year: 2016 - uzh · while both zevalin sand bexxar are composed of mabs...

Zurich Open Repository andArchiveUniversity of ZurichMain LibraryStrickhofstrasse 39CH-8057 Zurichwww.zora.uzh.ch

Year: 2016

New insights into the pretargeting approach to image and treat tumours

Patra, Malay ; Zarschler, Kristof ; Pietzsch, Hans-Jürgen ; Stephan, Holger ; Gasser, Gilles

Abstract: Tumour pretargeting is a promising strategy for cancer diagnosis and therapy allowing for therational use of long circulating, highly specific monoclonal antibodies (mAbs) for both non-invasive can-cer radioimmunodetection (RID) and radioimmunotherapy (RIT). In contrast to conventional RID/RITwhere the radionuclides and oncotropic vector molecules are delivered as presynthesised radioimmuno-conjugates, the pretargeting approach is a multistep procedure that temporarily separates targeting ofcertain tumour-associated antigens from delivery of diagnostic or therapeutic radionuclides. In princi-ple, unlabelled, highly tumour antigen specific mAb conjugates are, in a first step, administered into apatient. After injection, sufficient time is allowed for blood circulation, accumulation at the tumour siteand subsequent elimination of excess mAb conjugates from the body. The small fast-clearing radiola-belled effector molecules with a complementary functionality directed to the prelocalised mAb conjugatesare then administered in a second step. Due to its fast pharmacokinetics, the small effector moleculesreach the malignant tissue quickly and bind the local mAb conjugates. Thereby, corresponding radioim-munoconjugates are formed in vivo and, consequently, radiation doses are deposited mainly locally. Thisprocedure results in a much higher tumour/non-tumour (T/NT) ratio and is favourable for cancer di-agnosis and therapy as it substantially minimises the radiation damage to non-tumour cells of healthytissues. The pretargeting approach utilises specific non-covalent interactions (e.g. strept(avidin)/biotin)or covalent bond formations (e.g. inverse electron demand Diels–Alder reaction) between the tumourbound antibody and radiolabelled small molecules. This tutorial review descriptively presents this com-plex strategy, addresses the historical as well as recent preclinical and clinical advances and discusses theadvantages and disadvantages of different available variations.

DOI: https://doi.org/10.1039/c5cs00784d

Posted at the Zurich Open Repository and Archive, University of ZurichZORA URL: https://doi.org/10.5167/uzh-134643Journal ArticlePublished Version

The following work is licensed under a Creative Commons: Attribution 3.0 Unported (CC BY 3.0)License.

Originally published at:Patra, Malay; Zarschler, Kristof; Pietzsch, Hans-Jürgen; Stephan, Holger; Gasser, Gilles (2016). New in-sights into the pretargeting approach to image and treat tumours. Chemical Society reviews, 45(23):6415-6431.DOI: https://doi.org/10.1039/c5cs00784d

Chem Soc RevChemical Society Reviewswww.rsc.org/chemsocrev

ISSN 0306-0012

TUTORIAL REVIEW

Kristof Zarschler, Gilles Gasser et al.New insights into the pretargeting approach to image and treat tumours

Volume 45 Number 23 7 December 2016 Pages 6409–6658

This journal is©The Royal Society of Chemistry 2016 Chem. Soc. Rev., 2016, 45, 6415--6431 | 6415

Cite this: Chem. Soc. Rev., 2016,

45, 6415

New insights into the pretargeting approachto image and treat tumours

Malay Patra,a Kristof Zarschler,*b Hans-Jurgen Pietzsch,b Holger Stephanb andGilles Gasser*a

Tumour pretargeting is a promising strategy for cancer diagnosis and therapy allowing for the rational

use of long circulating, highly specific monoclonal antibodies (mAbs) for both non-invasive cancer

radioimmunodetection (RID) and radioimmunotherapy (RIT). In contrast to conventional RID/RIT where the

radionuclides and oncotropic vector molecules are delivered as presynthesised radioimmunoconjugates, the

pretargeting approach is a multistep procedure that temporarily separates targeting of certain tumour-

associated antigens from delivery of diagnostic or therapeutic radionuclides. In principle, unlabelled, highly

tumour antigen specific mAb conjugates are, in a first step, administered into a patient. After injection,

sufficient time is allowed for blood circulation, accumulation at the tumour site and subsequent elimination

of excess mAb conjugates from the body. The small fast-clearing radiolabelled effector molecules with a

complementary functionality directed to the prelocalised mAb conjugates are then administered in a second

step. Due to its fast pharmacokinetics, the small effector molecules reach the malignant tissue quickly and

bind the local mAb conjugates. Thereby, corresponding radioimmunoconjugates are formed in vivo and,

consequently, radiation doses are deposited mainly locally. This procedure results in a much higher tumour/

non-tumour (T/NT) ratio and is favourable for cancer diagnosis and therapy as it substantially minimises the

radiation damage to non-tumour cells of healthy tissues. The pretargeting approach utilises specific non-

covalent interactions (e.g. strept(avidin)/biotin) or covalent bond formations (e.g. inverse electron demand

Diels–Alder reaction) between the tumour bound antibody and radiolabelled small molecules. This tutorial

review descriptively presents this complex strategy, addresses the historical as well as recent preclinical and

clinical advances and discusses the advantages and disadvantages of different available variations.

Key learning points(1) Monoclonal antibodies (mAbs) are excellent tools to target tumour associated antigens, but the straightforward approach of utilising directly radiolabelled

mAbs for radioimmunodetection (RID) and radioimmunotherapy (RIT) of tumours is affected by their unfavourable biodistribution.

(2) The pretargeting approach is a multistep procedure that temporarily separates targeting of tumour associated antigens by mAb conjugates from delivery of

diagnostic or therapeutic radionuclides.

(3) The pretargeting approach can boost the tumour/non-tumour ratio and thus limits the radiation dose for healthy tissues.

(4) The pretargeting approach utilises specific non-covalent interactions (e.g. strept(avidin)/biotin or hybridisation of oligonucleotides) as well as covalent bond

formations (e.g. inverse electron demand Diels–Alder reaction) between the tumour bound antibody conjugates and radiolabelled small molecules.

1. Introduction

Early diagnoses, quantitative imaging, efficient treatment and

precise monitoring of anticancer therapy require extremely

sensitive detection methodologies such as the nuclear medicinal

imaging techniques single-photon emission computing tomo-

graphy (SPECT) and positron emission tomography (PET). In

addition to these, the use of a highly tumour-specific vehicle

labelled with a radionuclide as reporter for radioimmuno-

detection (RID) or warhead for radioimmunotherapy (RIT) is

essential to achieve high sensitivity and specificity.1 These

affine oncotropic vector molecules with appropriate pharmaco-

kinetic and pharmacodynamic properties become increas-

ingly important as an alternative to, or in combination with,

aDepartment of Chemistry, University of Zurich, Winterthurerstrasse 190,

CH-8057 Zurich, Switzerland. E-mail: [email protected];

Web: www.gassergroup.com; Tel: +41 44 635 46 30bHelmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmaceutical

Cancer Research, Bautzner Landstraße 400, D-01328 Dresden, Germany.

E-mail: [email protected]; Tel: +49 351 260 3678

Received 20th October 2015

DOI: 10.1039/c5cs00784d

www.rsc.org/chemsocrev

Chem Soc Rev

TUTORIAL REVIEW

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View Article OnlineView Journal | View Issue

6416 | Chem. Soc. Rev., 2016, 45, 6415--6431 This journal is©The Royal Society of Chemistry 2016

conventional treatments such as surgery, chemotherapy and

external radiotherapy.2

Monoclonal antibodies (mAbs) bind cognate antigens with

excellent specificity and thus represent indispensable mole-

cules for therapeutic and diagnostic applications. Since their

introduction more than 60 years ago, radiolabelled mAbs

have been extensively evaluated in preclinical and clinical

settings for delivering radioactivity to tumour sites to visua-

lise, characterise, and treat tumour malignancies including

primary and metastatic cancers.3 The potential of this so

called conventional RID/RIT is evidenced by the approval of

two radioimmunotherapeutic mAbs, namely ibritumomab

tiuxetan (Zevalins) and tositumomab (Bexxars), by the U.S.

Food and Drug Administration (FDA) for treatment of non-

Hodgkin’s lymphoma (NHL). While both Zevalins and Bexxars

are composed of mAbs directed against the CD20 antigen

which is overexpressed in B-cell lymphomas, they bear

different radionuclides. In the clinic, the 111In-labelled form

of Zevalins is used for diagnostic imaging or RID, whereas90Y-Zevalins is used for RIT. Bexxars was approved in

combination with the radioisotope 131I for treatment of

NHL.4 The principles, issues and challenges associated with

Malay Patra

Malay Patra completed his PhD

thesis with distinction (2011) in

the group of Prof. Nils Metzler-

Nolte, Ruhr University Bochum

(Germany). He was a recipient

of Max Planck fellowship for

international graduate students.

After his first postdoctoral stay in

the group of Prof. Gilles Gasser,

University of Zurich (Switzerland),

Malay joined the group of Prof.

Stephen J. Lippard, Massachussets

Institute of Technology (USA) for his

second postdoc (2013). Recently, he

came back to University of Zurich and started working as a Senior

Scientist in the group of Prof. Gilles Gasser. Throughout his research

career, Malay has been involved in the development of novel metal-

based antibacterial, antiparasitic and anticancer drug candidates.

Kristof Zarschler

After completing his university

studies of Biology at the Dresden

University of Technology, Germany,

in 2005, Kristof Zarschler started

his doctoral thesis at the Depart-

ment of NanoBiotechnology of the

University of Natural Resources and

Applied Life Sciences Vienna,

Austria. He received his PhD

degree with distinction in 2009

and came back to the Dresden

University of Technology, Germany,

where he joined the group of Prof.

Gerhard Rodel as research associate

(2010). Since 2011, Kristof works as research associate at the Institute of

Radiopharmaceutical Cancer Research of the Helmholtz-Zentrum

Dresden-Rossendorf, Germany. His research interests focus on the

application of antibody fragments as diagnostic and therapeutic

agents as well as targeting moieties for nanoparticles.

Hans-Jurgen Pietzsch

Hans-Jurgen Pietzsch studied

chemistry at Leipzig University

and received his PhD degree from

the Technical University Dresden

in 1990. Then, he worked as

senior researcher in the field of

Coordination and Radiopharma-

ceutical Chemistry particularly

on technetium and rhenium

chemistry. Since 2003, Hans-

Jurgen is leading the ‘‘Radio-

Theranostics’’ department at the

Institute of Radiopharmaceutical

Cancer Research of the Helmholtz-

Zentrum Dresden-Rossendorf. His research interests are the

development of radiometal complexes for diagnostic and

therapeutic applications.

Holger Stephan

Holger Stephan studied chemistry

and received his PhD degree

from the Technical University

Bergakademie Freiberg in 1989.

Then, Holger moved to the Tech-

nical University Dresden and

worked as senior researcher

together with Prof. Karsten Gloe

in the field of Supramolecular

Chemistry particularly on anion

recognition. Since 2005, he is

leading the ‘‘Nanoscalic Systems’’

group at the Institute of Radio-

pharmaceutical Cancer Research

of the Helmholtz-Zentrum Dresden-Rossendorf. Currently, Holger

is spokesperson of a Helmholtz Virtual Institute ‘‘Functional

nanomaterials for multimodal cancer imaging’’. His research

interests are the development of radiometal complexes, including

functionalised nanoparticles for therapeutic and diagnostic

applications, dendrimer chemistry, coordination chemistry, and

the thermodynamics of liquid two-phase systems.

Tutorial Review Chem Soc Rev

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RIT, radionuclides used in RIT as well as the latest progress

in this field have been the subject of numerous comprehen-

sive reviews,2,3,5,6 and the interested reader is directed to

these to obtain further information on that particular topic.

Despite their success in the clinic, the concept of utilising

radioimmunotherapeutic mAbs is afflicted with several

important shortcomings.4,7 mAbs are known for their high

stability and prolonged circulation in the blood, slow biodis-

tribution kinetics and slow clearance from the body. Due to

the poor tissue penetration abilities of radiolabelled mAbs, a

delayed clearance from the blood is required to achieve

higher accumulation of radioactivity in tumours. But, at

the same time, this prolong circulation appears as one of

the most important drawbacks of conventional RID/RIT.3

It causes undesired radiation burden in healthy tissues,

especially in the bone marrow, which is extremely sensitive

to radiation. This limits the opportunity of enhancing the

efficacy of therapy by increasing the dose of radiolabelled

mAbs. Additionally, slow clearance of radiolabelled mAbs

from blood and healthy tissues often results in a lower

tumour/non-tumour (T/NT) ratio and – for RID – it requires

extensive waiting periods before image acquisition. Rapid

clearance of radiolabelled mAbs from the blood and non-

targeted tissue is therefore beneficial in order to obtain a

higher T/NT ratio. As a consequence, the pharmacokinetic

properties of mAbs appear as the main dilemma in conven-

tional RID/RIT approach.7 Fortunately, however, several of

these shortcomings can be eliminated by implementing the

pretargeting strategy allowing for the rational use of long

circulating, high-affinity mAbs for both non-invasive cancer

RID and RIT.8

2. Detailed explanation of thepretargeting strategy

In contrast to conventional RID/RIT where the radionuclides

and oncotropic vector molecules are delivered as presynthesised

radioimmunoconjugates (Fig. 1A), the pretargeting strategy is

a multistep procedure that decouples targeting of tumour asso-

ciated antigens from radionuclide delivery (Fig. 1B). All pretarget-

ing systems share in principle two basic components: (i) a target

specific component, often a non-radioactive mAb conjugate

capable of binding a certain tumour antigen as well as (ii) a

small radiolabelled effector component. As schematically repre-

sented in Fig. 1, unlabelled, highly tumour specific mAb con-

jugates are in a first step administrated into a patient. After

injection, sufficient time, also referred to as pretargeting interval,

is allowed for blood circulation, accumulation at the tumour site

and subsequent elimination of excess mAb conjugates from the

body. Importantly, as the mAb conjugates themselves are not

radiolabelled, the pretargeting interval can be extended to several

days for optimal T/NT ratios without the risk of radiation

exposure to healthy tissues. At the time when the concentration

of mAb conjugates at the tumour site is at its peak and much

higher than in healthy tissues, small fast-clearing radiolabelled

effector molecules with a complementary functionality directed

to the prelocalised mAb conjugates are administered in a second

step. Due to its fast pharmacokinetics, the small effector mole-

cules reach the malignant tissue quickly and bind the local mAb

conjugates, thereby forming radioimmunoconjugates in vivo. The

excess effector molecules are rapidly excreted from the blood or

healthy tissues, resulting in a higher T/NT ratio which is favour-

able for diagnostics and therapy. A hallmark of all pretargeting

procedures therefore represents the minimised radiation expo-

sure of non-targeted tissue as well as limited haematological

toxicity.3 Following its initial demonstration by Reardan and his

team in 1985,9 the pretargeting strategy has become an area of

extensive research and extremely promising results have been

reported over the last decades.10,11

Although the pretargeting approach improves the T/NT ratio

significantly over conventional RID/RIT, it should be, however,

remembered that the pretargeting strategy comes with several

complexities which are absent in conventional RID/RIT.

Conventional targeting has two variables, namely the doses of

radiolabelled mAbs and time of detection. In general, provided

the mAb binding sites are unsaturated, the accumulation of

radioactivity in tumour is only slightly influenced by the doses

of the radiolabelled mAb but greatly varies with the detection

time. The optimisation of conventional RID/RIT in clinical

setting is therefore relatively easy. On the contrary, even the

simplest two-step pretargeting strategy involves four variables,

namely the doses of pretargeting mAb conjugates, the pretar-

geting interval, the doses of radiolabelled effector molecules

and the time of detection. The accumulation of radioactivity,

which is now not in the mAb but in the secondary effector

agent, is highly influenced by the changes in any of these four

variables. Additionally, there are three system-associated vari-

ables in the pretargeting approach (e.g. the mAb conjugates,

Gilles Gasser

Gilles Gasser completed a PhD

thesis in supramolecular chemistry

in the group of Helen Stoeckli-Evans

at the University of Neuchatel

(2004). After postdoctoral stays

at Monash University (Australia)

with Leone Spiccia in bioinorganic

chemistry and at the Ruhr-

University Bochum (Germany) with

Nils Metzler-Nolte in bioorgano-

metallic chemistry, Gilles started

his independent research career at

the University of Zurich, first as

Swiss National Science Foundation

(SNSF) Ambizione fellow (2010) and then as a SNSF Assistant Professor

(2011). Gilles is the recipient of several fellowships/awards including an

Alexander von Humboldt fellowship (2007), the Werner Prize from the

Swiss Chemical Society (2015), a European Research Council (ERC)

consolidator grant (2015) and the Thieme Chemistry Journals Award

(2016). Gilles’ research interests lie in the use of metal complexes in

medicine and chemical biology.

Chem Soc Rev Tutorial Review

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the radiolabelled secondary effector agent and the type of tumour)

compared to only two in conventional RID/RIT (e.g. the radio-

labelled vector molecule and the type of tumour). Complexity

further increases with increasing the number of steps, making

the optimisation very difficult in clinical settings. The optimisa-

tion of a pretargeting system is carried out experimentally in

practice by varying different variables using trial and error

methods. The optimised parameters are also not broadly applic-

able to any system, but strictly specific to a particular pretargeting

system. Therefore, optimisation of variables is required for each

pretargeting system undergoing clinical trial and such optimisa-

tion is not very practical. Liu and Hnatowich proposed a semi-

empirical model for pretargeting RID/RIT, which is capable of

estimating how the optimal tumour accumulation and T/NT ratio

is influenced by the different combination of variables. The

model is based on several quantitative equations relating the

dependence of different pretargeting variables on the outcome.

For the details of this model, from the design to the applicability,

readers are directed to the excellent review published by the

pioneer of this model.12 This semiempirical model has thus far

been applied only to a MORFs/cMORFs (MORF = phospho-

diamidate morpholino oligomers) pretargeting system. More

and more experimental data on other pretargeting systems is

required to predict the true applicability of this semiempirical

model on the optimisation of pretargeting variables without an

exhaustive trial and error method.

All the pretargeting systems introduced to date can be

classified into two broad categories depending on the interactions

between the pretargeted mAb conjugates and the radiolabelled

small effector molecules: (i) non-covalent high affinity interaction

and (ii) covalent bond formation using bioorthogonal chemistry.

These two categories are presented in detail below.

3. Non-covalent high affinityinteractions3.1 Recognition systems based on bispecific antibodies

The initial pretargeting concept described more than 30 years

ago pursued the strategy to apply bispecific (bs) monoclonal

antibodies (mAbs) capable of binding both tumour antigens as

well as radiolabelled chelates (Fig. 2).11

The first generation of bs mAbs-based pretargeting systems

originally involved bs mAbs consisting of a single anti-tumour

Fab fragment that was chemically conjugated to a single anti-

chelate Fab fragment resulting in both monovalent binding to

the tumour antigen as well as to the radiometal–chelate

complex (Fig. 2A).7 In the initial preclinical and clinical studies

Fig. 1 Schematic presentation of conventional radioimmunodetection (RID) and radioimmunotherapy (RIT) with directly radiolabelled mAbs (A) andpretargeting procedure using mAb conjugates and small radiolabelled effector molecules (B). Conventional RID/RIT uses radionuclide-carrying tumourspecific mAbs with long blood retention times resulting in detrimental radiation exposure of healthy tissues (A). In contrast, the pretargeting approachstarts with the intravenous administration of unlabelled, highly tumour specific mAb conjugates into the patient. After accumulation of the mAbconjugates at the tumour site and clearance from non-targeted tissues, small fast-clearing radiolabelled effector molecules are injected that bind to theprelocalised mAb conjugates at the tumour site (B).


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using this pretargeting system, Fab fragments against the carcino-

embryonic antigen (CEA) were combined with Fab fragments

binding to DTPA. Since antibodies bound to CEA do not readily

internalise and thus remain accessible at the surface of tumour

cells, this particular tumour antigen is especially suitable for

pretargeting. Taking full advantage of this behaviour, an extended

time period for clearance of the bs mAbs of up to eight days can

be allowed before injection of the fast-clearing radiometal–chelate

complexes. Compared to directly radiolabelled but long circulating

anti-CEA IgG antibodies, improved tumour-to-blood and tumour-

to-liver ratios were obtained using the two-step pretargeting

method due to the fast clearance of the radiolabelled chelates.13

Although this pretargeting system was successfully tested in

patients with primary colorectal cancer,14 the radiolabelled

chelates were rapidly released from the tumour site due to their

weak monovalent interactions with the pretargeted bs mAbs.

Higher tumour uptake and retention were achieved by gradually

improving the different components of the monovalent binding

system. The second generation of bs mAbs based pretargeting

systems deployed short peptides linking two radiometal–chelate

complexes instead of monomeric radiolabelled chelates (Fig. 2B).7

These bivalent probes interact with the anti-chelate Fab fragment

of the prelocalised bs mAbs with enhanced avidity resulting

in enhanced tumoural uptake and retention.11 The group of

Kraeber-Bodere intensively investigated this second generation

pretargeting procedure regarding the treatment of CEA-

producing medullary thyroid carcinoma (MTC) in several pre-

clinical as well as clinical studies10 and critically compared the

toxicity and efficacy of pretargeted RIT with those of conven-

tional RIT.15

The first as well as second generation of bs mAbs-based

pretargeting systems utilised anti-chelate antibodies with an

exceptional specificity for a certain radiometal–chelate complex,

e.g. 111In-loaded DTPA. The major drawback of these approaches

was that the binding affinity dropped substantially, when other

radiometals were complexed by the same chelator as observed for90Y– or 177Lu–DTPA. This limited flexibility necessitated, in the

worst case, the generation of new antibodies with high affinity for

the desired radiometal–chelate pairs, e.g. directed against 90Y– or177Lu–DOTA, in order to expand these pretargeting systems to a

wide variety of radionuclides.13

To circumvent this limitation, the anti-chelate Fab fragment

of the bs mAbs was replaced by a Fab fragment binding to an

inert compound composed of histamine-succinyl-glycine (HSG)

with nanomolar affinity. An additional small peptide core of

three to four D-amino acids serves on one hand as a metabo-

lically stable scaffold to link two HSG moieties resulting in

a bivalent HSG-peptide. On the other, the peptide can be

modified in different ways to attach diverse ligands capable

of binding the radionuclide of choice. This included the

(3-thiosemicarbazonyl)glyoxylcysteinyl (Tscg-Cys) moiety for

complexing of 99mTc and 188Re as well as DOTA for chelating111In, 68Ga, 177Lu and 90Y for diagnostic or therapeutic applications,

respectively. Furthermore, incorporation of a tyrosine residue into

the peptide sequence also allowed for radioiodination.11

For the third generation of bs mAbs-based pretargeting

systems, bs mAbs with two binding sites for the tumour antigen

and one for the HSG-peptide were prepared by a modular

approach termed Dock-and-Lock procedure (Fig. 2C).7 This

innovative and sophisticated system stably joins one Fab frag-

ment of an anti-HSG antibody and two Fab fragments of an

anti-tumour antibody (tri-Fab bs mAbs) in a manner to allow

for bivalent binding to the tumour antigen and monovalent

binding of the radiolabelled HSG-peptide. Due to its bivalency,

a single HSG-peptide can crosslink two nearby tri-Fab bs mAbs

at the surface of tumour cells leading to the formation of a

stable complex. For detailed information on the Dock-and-Lock

method the interested reader is directed to an excellent review

of Rossi et al.16

Several clinical trials for identifying optimal treatment para-

meters and assessing safety as well as tolerance of the third

generation of bs mAbs-based pretargeting system in humans

were started in patients with metastatic colorectal and lung

cancer as well as medullary thyroid carcinoma, respectively.

The corresponding results of these optimisation studies using

anti-CEA tri-Fab bs mAbs in combination with 111In and 68Ga

Fig. 2 Development of bs mAbs-based pretargeting. The first generationof bs mAbs-based pretargeting systems involved bs mAbs consisting of asingle anti-tumour Fab fragment chemically conjugated to a single anti-chelate Fab fragment resulting in monovalent binding to the tumourantigen as well as to the chelate (A). The second generation of bs mAbs-based pretargeting systems utilised bivalent peptide-linked chelates possiblybridging two adjacent bs mAbs (B). Bs mAbs with two binding sites for thetumour antigen and one for the HSG-peptide (tri-Fab) were prepared in thethird generation bs mAbs based pretargeting system. The radiolabelledeffector molecule was modified to a bivalent HSG-peptide (C).


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(for imaging) or 177Lu (for therapy) labelled bivalent HSG-

peptides were reported recently.17–20 Points of particular note

are in this context the fast and selective tumour visualisation

within hours after injection of the radiolabelled di-HSG-peptides

subsequently to pretargeting with the anti-CEA tri-Fab bs mAbs

as well as fast clearance from non-targeted tissues and organs

resulting in high tumour-to-normal tissue ratios. Moreover,

injected radiation doses of 177Lu ranging from 2.5–7.4 GBq

resulted only in limited haematological toxicity, and no signs or

symptoms of renal toxicity were observed.

Of possible importance for repeated administration of bs

mAbs is the appearance of immune responses in a number

of patients such as infusion reactions and the formation of

human antibodies against the tri-Fab bs mAbs although huma-

nised recombinant antibody constructs were injected. The

presence of such antibodies during a therapy cycle may affect

the pharmacokinetics of the bs mAbs and influence the quality

of tumour targeting, whereas the former adverse event is

manageable by appropriate anti-allergic premedication with

antihistamines and corticosteroids injected.17,19

In summary, the bs mAbs-based pretargeting systems have

been intensively investigated in preclinical as well as several

clinical studies for RID and RIT of cancer and documented in

detail. These systems appear on one hand suitable for RID by

immuno-PET, when the effector molecules are labelled with

short half-life positron-emitters such as 68Ga or 64Cu. On the

other hand, ongoing clinical trials will analyse their performance

for RIT as promising preclinical studies have demonstrated the

very fast delivery of high radiation doses to tumour tissues with

limited exposure of non-targeted tissues.

3.2 Avidin/streptavidin–biotin recognition system

The avidin/streptavidin–biotin system relies on the very high

affinity of (strept)avidin for biotin and was initially introduced

by Donald J. Hnatowich and members of his group.21 Up to four

biotin molecules (also known as vitamin B7, vitamin H or

coenzyme R) can bind to the B66 kDa glycoprotein avidin with

an dissociation constant Kd of 10�15 M. This is one of the

strongest known non-covalent interactions, whereas their asso-

ciation is rapid, and, once formed, this complex is stable to

extremes of pH and in the presence of denaturing agents or

organic solvents. Of note, the non-glycosylated bacterial analo-

gue of avidin, streptavidin (B56 kDa), is interchangeable with

avidin in in vitro studies. However, for in vivo applications, the

difference in glycosylation between the two proteins makes

them suitable for different purposes as naturally glycosylated

avidin, in contrast to non-glycosylated streptavidin, is rapidly

sequestered by the liver.22 Worthy of note, streptavidin was

reported to show less non-specific binding to tissues compared

to avidin.21 The avidin/streptavidin–biotin system requires

either the conjugation of (strept)avidin to mAbs or the generation

of biotinylated mAbs. Both configurations have been reported

frequently and differ in their application regime.22

The two-step method starts with the intravenous injection of

anti-tumour streptavidin mAb conjugates (Fig. 3A). Their high

molecular weight (B210 kDa) results in a slow blood clearance

and necessitates the removal of excess streptavidin mAb con-

jugates from the blood pool by administration of a clearing

agent prior to the injection of the radiolabelled biotin molecules.

Once the concentration of the streptavidin mAb conjugates peaks

at the tumour site, the clearing agent (e.g. biotinylated galactose-

conjugated human serum albumin) is given to transport circulating

streptavidinmAb conjugates to the liver, where they aremetabolised

and withdrawn from circulation.22

In contrast, the three-step method of the avidin/streptavidin–

biotin system uses biotinylated antitumour mAbs and glycosy-

lated avidin as clearing agent, which is given once the biotinylated

mAbs have achieved maximum tumour uptake (Fig. 3B). After the

circulating complex composed of avidin and biotinylated mAbs is

transported to the liver, unlabelled streptavidin is administered.

Upon reaching the malignant tissue, these multivalent proteins

strongly bind to the prelocalised biotinylated mAbs. Finally, radio-

labelled biotin molecules are injected and interact with tumour-

bound complexes of streptavidin and biotinylated mAbs.22

Fig. 3 Schematic representation illustrating the two-step (A) and three-step (B) method of the avidin/streptavidin–biotin pretargeting system. Thetwo-step method includes the injection of anti-tumour streptavidin mAbconjugates to pretarget the malignant tissue. Once the streptavidin mAbconjugates have achieved maximum tumour uptake, a clearing agent isapplied to remove the streptavidin mAb conjugates from the bloodcirculation before radiolabelled biotin is given (A). The three-step methodstarts with the administration of biotinylated anti-tumour mAbs localisingto the malignant tissue. After the injection of the clearing agent, unlabelledstreptavidin is given to bind the pre-localised biotinylated mAbs at thetumour site. Finally, radiolabelled biotin is injected to interact with tumour-bound complexes of streptavidin and biotinylated mAbs (B).


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In their seminal paper, Hnatowich and co-workers investi-

gated the in vivo application of mAbs linked to (strept)avidin

or biotin followed by the administration of 111In-labelled

(strept)avidin or biotin. They could demonstrate that the intra-

venous administration at first of avidin mAb conjugates followed

by radiolabelled biotin was more beneficial than the reverse

procedure (i.e. injection of biotin mAb conjugates followed by

radiolabelled streptavidin). Indeed, the authors observed an

unfavourable slow blood clearance of 111In-labelled streptavidin

and its accumulation in normal tissue. Promisingly, using

protein-A conjugated beads to simulate tumours, Hnatowich

and his team showed that T/NT ratios were increased by more

than two orders of magnitude over the conventional use of a

single intravenous administration with radiolabelled mAb.21

Following this pioneering work, several research groups have

employed this in vivo recognition system in view of imaging

and treatment of tumours using different radionuclides (e.g. 111In,153Sm, 90Y, 177Lu and 211At).23,24

Due to space constrains, it is impossible to report individually

each study on this recognition system. Thus, we have decided,

in the next paragraph, to only highlight the results of clinical

studies that have utilised the avidin/streptavidin–biotin system

in pretargeting. For detailed information on the application

of this recognition system in cancer diagnosis and therapy,

the interested reader is directed to two excellent reviews by

Lesch et al. and Stoldt et al., respectively, as well as references

therein.23,24

To the best of our knowledge, Hnatowich and his team were

the first researchers to report a clinical study. Specifically,

streptavidin was conjugated to mouse mAbs directed against

a component of human milk fat globule membranes and

reacting strongly with a range of neoplasms. These streptavidin

mAb conjugates were intravenously administered to ten patients

with documented squamous cell carcinoma of the lung. Two to

three days later, 111In-labelled biotin was injected and patients

were imaged 2 h after the administration for detection of lung

cancer lesions. Importantly, no toxicity for the patients at the

doses tested was evidenced in this study. Background radio-

activity levels were found to be low in liver and kidneys as well as

in other normal tissues and blood. In fact, the radioactivity levels

in liver and kidney were improved by at least one order of

magnitude and by a factor of five, respectively, compared to

conventional approach using directly radiolabelled mAbs.23

Shortly afterwards, the Milanese group of Giovanni Paganelli

and co-workers described two diagnostic clinical trials on the

two-step as well as three-step method of the avidin/streptavidin–

biotin system. The former study was conducted on ovarian

cancer patients and the latter focussed on carcinoembryonic

antigen-positive patients. Both trials utilised biotinylated mAbs

and provided a deep insight into pharmacokinetics, biodistribu-

tion and tumour localisation. In particular, their elaborated

three-step pretargeting protocol avoids the issues of slow blood

clearance of radiolabelled (strept)avidin and its accumulation in

normal tissues. According to the authors, the advantages of this

three-step method over other pretargeting approaches or direct

radiolabelling of mAbs are manifold. Firstly, the small size of the

radiolabelled biotin molecules ensures their rapid renal elimina-

tion and allows for diagnostic imaging already shortly after

injection. As the retention time in the body is thereby reduced,

the radiation exposure of liver and bone marrow is minimised.

Secondly, circulating biotinylated antitumour mAbs interact with

the clearing agent avidin and both molecules are metabolised

by the mononuclear phagocyte system of the liver as non-

radioactive complex leading to background reduction. Thirdly,

the mild reaction conditions applied for biotinylation of the

mAbs preserve their immunoreactivity. As the mAbs are not

directly labelled, also their radiolysis is prevented. Fourthly,

signal amplification can potentially be achieved through the

multivalency of streptavidin. In theory, multiple streptavidin

molecules can bind to prelocalised biotinylated mAbs and

multiple radiolabelled biotin molecules can finally interact

with tumour-bound complexes of streptavidin and biotinylated

mAbs. And fifthly, the three-step pretargeting protocol resulted

in substantial improvement of T/NT ratios within a few hours

after injection of the radiolabelled effector molecules expanding

the range of suitable radionuclides.24

Finally, the authors mentioned that their technique presents

a few drawbacks, namely repeated injections at defined time

intervals and the immunogenicity of avidin. The latter, very

important point will be discussed later in this section of the

review.

The same research group conducted in the following years

several diagnostic and therapeutic studies on patients with

different cancer entities including head and neck cancer as

well as glioblastoma.13,25 Of note, the delivery of therapeutic

radionuclides, in particular 90Y, by the three-step method of the

avidin/streptavidin–biotin system in patients with high-grade

glioma resulted in tumour mass reduction and survival benefit.

In comparison to conventional RIT using directly labelled

mAbs, this pretargeting approach indeed allows for the admini-

stration of high 90Y activities enabling delivery of a high dose to

the tumour and sparing red marrow and other normal organs.13

At this stage, although promising clinical trials were indeed

undertaken, as already discussed above, it is important to high-

light that the avidin/streptavidin–biotin recognition system still

presents several drawbacks. As mentioned by Goldenberg et al.,

who debated with Giovanni Paganelli and Marco Chinol on the

viability of this recognition system in pretargeting, immuno-

genicity is problematic.26,27 The immune system of patients

recognises the foreign biotin and produces antibodies against

the biotin after the first injection. Upon a second application, the

biotin can initiate a severe immune reaction. Also, the production

of the antibody–protein conjugate is relatively complicated and

its diffusion/adsorption is relatively slow due to its relatively

large size.

3.3 Recognition systems based on in vivo hybridisation

of synthetic complementary oligonucleotides

DNA as well as RNA oligonucleotides of a certain sequence

rapidly bind their complementary strands with a very high

specificity and affinity. However, natural occurring nucleic

acids are highly susceptible to nuclease-mediated hydrolysis


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and possess therefore an insufficient in vivo stability. For that

reason, several synthetic non-immunogenic DNA analogues with

excellent chemical and metabolic stability have been developed

as recognition systems for tumour pretargeting including peptide

nucleic acids, phosphorodiamidate morpholino oligomer and

mirror-imaged oligonucleotides (Fig. 4).

3.3.1 Morpholino nucleic acids as recognition units. Phos-

phordiamidate morpholino oligomers (morpholinos or MORFs)

are a distinct class of non-ionic oligonucleotide analogues. In

these synthetic molecules, a morpholine-phosphorodiamidate

backbone replaces the ribofuranose-phosphodiester linkage

present in natural DNA and RNA (Fig. 4B). This backbone

makes morpholinos water-soluble and resistant against enzymatic

degradation by nucleases. Despite their altered backbone, mor-

pholinos bind to their complementary nucleic acid sequences

by Watson–Crick base-pairing. Advantageously, while the

ribofuranose-phosphate backbone in DNA and RNA is chiral,

there is no such complexity in hybridisation of morpholino

oligomers.28 Pioneering work in the application of morpholinos

to the field of radiopharmaceutical was performed by Hnatowich

and co-workers, who demonstrated the first proof-of concept use

of tumour pretargeting in mice using 18mer MORF and its 99mTc-

labelled 18mer complementary MORF (cMORF).28 The underlying

principle is illustrated in Fig. 5.

The anti-CEA antibody MN14, conjugated with amine-modified

MORF, hybridises with 99mTc-labelled MAG3–cMORF-conjugate

to achieve pretargeting in tumour-bearing mice. MAG3 (mercapto-

acetyl triglycine) is a tripeptide chelator especially designed for

stable binding of 99mTc in its oxidation state +5. Biodistribution

studies demonstrated good radioactivity uptake in tumour.

However, the 99mTc-labelled cMORF effector molecule also

accumulated in the kidneys and intestines. This accumulation

may be problematic for tumour imaging (or tumour therapy)

within the abdomen. To overcome this obstacle, changing the

radionuclide, and consequently, the chelator can be considered.

It is well-documented that radiolabelling with different radio-

nuclides via different chelators may alter the pharmacokinetics

of even large molecules such as peptides or antibodies. Conse-

quently, Hnatowich et al. compared pharmacokinetics of99mTc–MAG3–cMORF and 111In–DTPA–cMORF in normal mice

in a further study.29 Therein tumour-bearing mice were

pretargeted with the appropriate MORF-antibody. 48 h later99mTc–MAG3–cMORF and 111In–DTPA–cMORF, respectively,

were then injected and the mice were reimaged. The authors

clearly demonstrated that the biodistribution of the cMORF

effector was influenced by the appropriate chelate unit both in

normal and pretargeted tumour-bearing mice. As illustrated in

Fig. 6, accumulation of 111In–DTPA–cMORF in the intestines

was minimal compared with the intestinal accumulation of99mTc–MAG3–cMORF.

After demonstrating the rapid tumour accumulation and

normal tissue clearance of the 99mTc-labelled MAG3–cMORF/

MN14–MORF pretargeting system, Hnatowich et al. reported

the first preclinical mouse-model of MORF pretargeting for RIT

using the b�-emitting radionuclide 188Re. 188Re was selected as

the therapeutic radionuclide because of its attractive properties

for radiotherapy. It is a generator-based nuclide with a half-life

of 17 h and an average b-energy of about 2 MeV. A g-radiation

component of 0.155 MeV allows for SPECT imaging. Further-

more, diagnostic 99mTc and therapeutic 188Re form a so-called

‘‘matched pair’’ of radionuclides showing similar coordination

properties. This similarity often results in radiolabelled agents

with similar, if not identical, properties.

The proof-of-principle study was performed using 188Re–MAG3–

cMORF/MN14–MORF with 14.8 GBq 188Re per mouse and three

control groups (untreated mouse, injection of MN14–MORF alone,

Fig. 4 Schematic drawing comparing deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) with peptide nucleic acids (PNA, A), phosphorodiamidatemorpholino oligomer (B) and mirror-imaged oligonucleotides (C).


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injection of 188Re–MAG3–cMORF alone) and resulted in fast accu-

mulation of 188Re in tumour accompanied by rapid clearance from

normal tissues. The tumour accumulation was about four times

higher than in the kidney and blood, which were the healthy

tissues with highest radioactivity uptake. Tumour growth in

the study group ceased one day after radioactivity injection,

whereas tumours continued to grow at the same rate among the

three control groups. The therapeutic success was confirmed by

measuring the tumour size. On the fifth day after treatment, the

average net tumour weight was significantly lower for the study

group compared with the three control groups.30

A second preclinical study treating LS174T-tumour bearing

mice using 188Re–MAG3–cMORF and an MORF-modified

antiTAG-72 mAb CC49 was published by Hnatowich et al. four

years later. As shown by SPECT/CT, the radioactivity uptake was

highly tumour specific for the two time points given: 3.5 and

10 h after injection (Fig. 7). Except for the intense urine radio-

activity at 3.5 h, the radioactivity is essentially restricted to

tumour at both time points. Tumour growth was inhibited for

the treated mice and at the highest 188Re dose, a complete but

temporary tumour remission was evident in three out of five

animals. Histological examination of tissues from these animals

showed no evidence of cytotoxicity to normal tissues but obvious

radiation damage to the tumour.31

From a radiopharmaceutical point of view, the trivalent

metals In3+ and Y3+ constitute another ‘‘matched pair’’ of

radionuclides. 90Y is considered as an alternative to 188Re with

comparable b�-energy but of longer half-life (2.2 MeV, 64 h)

that can potentially improve radiotherapy effectiveness. 90Y will

Fig. 5 Principle of using oligomeric nucleic acids and synthetic DNA analogues for tumour pretargeting. In this pretargeting approach, an unlabelled,highly specific mAb–oligonucleotide conjugate has sufficient time to target a tumour before administration of a small fast-clearing radiolabelledcomplementary oligonucleotide that hybridises with the pre-localised mAb–oligonucleotide conjugate at the tumour site (A). Changing the radionuclideincluding the chelator may alter the pharmacokinetics of the effector molecule (B).

Fig. 6 Anterior projections of the SPECT/CT fused acquisitions obtainedat two time points in normal mice receiving either 99mTc–cMORF (leftpanel) or 111In–cMORF (right panel). Bladder 99mTc and 111In radioactivitywere digitally removed in all projections, as was the 111In contamination onthe animal’s coat in the 68 min 111In projection. Adapted with permissionfrom ref. 29, copyright Springer.


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provide higher tumour-to-organ absorbed radiation dose because

a larger portion of the circulating 90Y will be cleared without

depositing its energy in the normal tissues.

Based on the pretargeting study described above using188Re–MAG3–cMORF, Hnatowich and co-workers performed a

therapeutic pretargeting experiment using 90Y-labelled cMORF

sequences.32 Due to the different coordination chemistry of

yttrium compared to rhenium, the chelator MAG3 was substi-

tuted by the macrocyclic ligand DOTA forming a 90Y–DOTA–

cMORF/MORF–CC49 pretargeting system. The biodistribution

was measured in both normal and LS174T-tumour bearing

mice, and the pharmacokinetics of the 90Y–DOTA–cMORF

in both the normal the pretargeted mice was comparable to

that observed previously with 188Re–MAG3–cMORFs. While the90Y–DOTA–cMORF cleared rapidly from normal tissues, tumour

clearance was very slow and tumour radioactivity accumulation

was constant for at least seven days allowing for the T/B ratio to

increase linearly from 6 to 25 over this period. Therefore, by

extrapolation, normal tissue toxicities following administration

of therapeutic doses of 90Y may be comparable to that observed

for 188Re in which the T/B ratio increased from 5 to 20 over a

7 day period. Furthermore, due to the longer physical half-life,

the tumour-to-non-tumour absorbed radiation dose ratios were

improved in most organs and especially in blood.

Of note, the authors clearly showed that both the pharmaco-

kinetics and pretargeting behaviour of the cMORF effector is

generally not influenced by the nature of the chelator and the

radionuclide using 188Re–MAG3–cMORF and 90Y–DOTA–cMORF.

In summary, the preclinical studies performed by the group

of Hnatowich have clearly demonstrated the suitability and

effectiveness of the morpholino-based pretargeting system, with

high T/B ratios but no clinical trials have been initiated yet.

3.3.2 Peptide nucleic acid as recognition units. In 1991,

Nielsen et al. reported on the synthesis and characterisation of

thymine-based oligonucleotides where the deoxyribose phos-

phate backbone of DNA was replaced by a polyamide backbone.

They named it polyamide nucleic acids. Meanwhile, this class

of artificial oligonucleotides is well-known as peptide nucleic

acids (PNAs).33 PNAs contain repetitive N-(2-aminoethyl)-glycine

units attached to nucleobases via a methylcarbonyl linker to give

a flexible, non-chiral and non-charged pseudo-peptide polyamide

backbone (Fig. 4A). Due to the lack of electrostatic repulsion,

PNAs rapidly hybridise to form more stable duplexes than

PNA/DNA and in particular DNA/DNA.33 These oligonucleotides

have been primarily developed as recognition units for double-

stranded DNA. PNAs are characterised by an excellent chemical

and metabolic stability. They are almost inert towards degrada-

tion in vivo and can be synthetically modified to modulate their

intrinsic physico-chemical properties. For example, the attachment

of appropriate spacer elements and specific building blocks, such

as targeting units and solubility enhancers, to both the C- and

N-terminus of PNAs allows for a broad variation of the pharmaco-

logic properties of PNA bioconjugates.33 Of particular interest is

the linkage ofmetal complexes to PNAs. In thismanner, numerous

new applications have been explored.34 Metal-containing PNAs

play an important role in the development of artificial enzymes,

biosensors, DNA-templated metal catalysis, oxidative cross-linkers

to a DNA target. Moreover, metal–PNA conjugates are effective

modulators of PNA/DNA hybrid stability as well as find use as

radioactive probes. With regard to the latter, 99mTc, 111In- and64Cu-labelled PNAs have been evaluated for brain, breast, small

lymphotic lymphoma and pancreatic cancer imaging.35

The potential of PNAs as an efficient pretargeting system has

been explored by the group of Donald J. Hnatowich for the first

time in 1997.36 A 15-mer PNA oligomer equipped with MAG3 as

chelator was radiolabelled with 99mTc to show rapid renal

clearance in mice. In vivo hybridisation was confirmed using

polymeric beads transplanted into mouse thighs that contain the

complementary PNA strand. The highest amount of radioactivity was

found after 23 h in the transplanted beads. At this time, radioactivity

could only be detected in beads, kidneys and bladder.36

We recently described active tumour pretargeting with PNA

bioconjugates as complementary system. More specifically, we

modified on one hand epidermal growth factor receptor (EGFR)

Fig. 7 Anterior, left lateral and posterior projections for each SPECT/CT acquisition of one pretargeted mouse imaged at 3.5 h (A) and 10 h (B) afterinjection of 188Re–cMORF. Adapted with permission from ref. 31, copyright Taylor & Francis Ltd.


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specific therapeutic mAbs (cetuximab) with PNA oligomers

consisting of 17 bases. On the other, we attached dipicolyl (DPA:

N,N-bis(2-picolyl)amine) chelators to complementary PEGylated

PNA oligomers with optimised pharmacological properties.37 It is

worth highlighting that PEGylation of complementary effector

PNAs led to an increased blood residence time without substan-

tially influencing their hybridisation properties. Furthermore, these

PNA conjugates rapidly cleared from the body via the kidneys and

already 1 h postinjection, radioactivity was mostly detected in the

kidneys and bladder (Fig. 8A). Very importantly, rapid and efficient

hybridisation of the complementary 99mTc-labelled effector PNAs

with pretargeted mAb–PNA conjugates was observed in in vivo

experiments using EGFR-positive tumour xenografts (Fig. 8B

and C). This active tumour pretargeting approach permits a clear

visualisation of the tumour just 1 h postinjection of the radio-

labelled effector, achieving tumour-to-muscle ratios 48.37

Altogether, PNAs are promising candidates for further

preclinical studies. In this perspective, it is advantageous that

PNA derivatives can be produced with a PNA synthesiser,

allowing for rapid access to a broad range of PNA constructs

in an appropriate amount.

3.3.3 Use of mirror-imaged oligonucleotides as recognition

units. Mirror-imaged oligonucleotides are synthetic RNA or

DNA oligonucleotides based on the non-natural enantiomeric

forms L-ribofuranose or L-deoxyribofuranose, respectively (Fig. 4C).

Due to their L-configuration these compounds are resistant against

enzymatic degradation by nucleases and therefore stable in a

biological environment. Moreover, L-oligonucleotides do not

hybridise with nucleic acids of natural configuration.38 The

rapid hybridisation of complementary mirror-image oligo-

nucleotides, the lack of natural hybridisation targets as well

as their high metabolic stability makes L-oligonucleotides a

promising complementary system for pretargeting approaches.

Schlesinger et al. described the synthesis and radiopharmaco-

logical characterisation of 68Ga- and 86Y-labelled DOTA–12mer-L-

RNA conjugates as molecular probes for PET.39 Biodistribution

studies of the 86Y-labelled 12mer-L-RNA oligonucleotide in

healthy male Wistar rats showed very fast renal elimination

and low retention of the compound in tissues and organs. The

same group investigated the influence of the 86Y–DOTA chelate

moiety on the biodistribution of the 86Y–DOTA–12mer-L-RNA

conjugate in healthy rats.40

The very fast blood clearance combined with high kidney

accumulation of radiolabelled L-oligonucleotides requires opti-

misation to design an efficient pretargeting system. The objective

was to minimise the kidney uptake and non-specific accumulation,

and to increase the blood availability of radiolabelled L-oligo-

nucleotides. Introduction of high-molecular-weight polyethylene

glycol (PEG) moieties into a pharmacologically active substance

is a well-established tool to influence its pharmacokinetics.

Therefore, Foerster et al. investigated the impact of increasing

(2, 5, 10 and 20 kDa) PEG sizes of 68Ga- and 64Cu-labelled

L-oligonucleotide–PEG conjugates on their distribution and

PET kinetics in Wistar rats.41 It could be demonstrated that

tuning of pharmacokinetics is possible by successive PEGylation

resulting in a series of suitable L-oligonucleotides with reduced

kidney uptake, favourable half-life of circulation and very low

unspecific tissue accumulations. The introduction of NOTA or

DOTA as chelating moieties had no influence on the pharmaco-

kinetics and allows radiolabelling with various metallic radio-

nuclides for theranostic applications.

To conclude, the use of mirror-imaged oligonucleotides as

recognition units in pretargeting systems showed great promise

but is only in its infancy. Further studies are definitely required

concerning dosimetry and proof-of-feasibility in vivo.

4. Covalent bond formation usingbioorthogonal chemistry

All pretargeting systems evaluated in the past are promising

but each of them has specific advantages as well as limitations.

Fig. 8 SPECT/CT maximum intensity projection images of [99mTc](Tc-Dpa)–(Cys-PEG10kDa)–PNA in murine A431 tumour xenograft (NMRI nu/nu mice;tumour located at right thigh). (A) 1 h postinjection of [99mTc](Tc-Dpa)–(Cys-PEG10kDa)–PNAwithout preinjection of (NOTA)3-C225-Cys-c-PNA. (B) 1 h postinjection of radiotracer; (NOTA)3-C225-Cys-c-PNA was administered 24 hbefore injection of radiotracer. (C) 20 h postinjection of radiotracer; (NOTA)3-C225-Cys-c-PNA was administered 24 h before injection of radiotracer.Adapted with permission from ref. 37, copyright Royal Society of Chemistry.


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For example, the streptavidin and biotin couple offers the high-

est binding affinity and was found to be one of the promising

pretargeting systems thus far in clinical settings. However, as

discussed in more detail above, the system is not free from

limitations, such as the induction of immunogenicity caused

by the administration of streptavidin. Moreover, complexity in

engineering, the high cost of production of streptavidin or of the

biotin-attached mAb, and the perturbation of antibody affinity

while attached to streptavidin are additional drawbacks of this

system.42

In the recent past, bioorthogonal reactions have been tested

as an alternative to non-covalent interactions in the pretargeting

strategy. These chemical procedures are relatively straightforward,

less likely to induce immunogenicity and rely on covalent bond

formation between the tumour bound pretargeted mAbs and

the radiolabelled effectors. In this context, it is important to

note that a chemical reaction needs to meet certain criteria to

become truly bioorthogonal and to be useful for this purpose.

Those criteria are, for example, a high stability of the reaction

components in aqueous/biological media, a fast ligation kinetics

even at very low reagents concentration, an occurrence without

external reagent/catalyst, a selective reaction between the com-

ponents and no cross reaction with biological reactants in

cellular environment, a good yield and no generation of toxic

by-products.43 A few click chemistry reactions, namely, the

Staudinger ligation, the strain promoted alkyne–azide cyclo-

addition (SPAAC) and the inverse electron demand Diels–Alder

reaction (IEDAA), satisfy most of the above criteria and have

been evaluated for their potential in pretargeting in cultured

cells as well as in mice (Fig. 9). The progress made especially

with tumour pretargeting using bioorthogonal chemistry was

summarised in a few excellent reviews published recently.42,44,45

We therefore decided to limit our discussion to brief overviews of

the historical developments and a few specific landmark studies

Fig. 9 Schematic drawing of target specific and small fast-clearing radiolabelled effector components as well as their corresponding reaction productsdescribed in bioorthogonal chemistry based tumour-pretargeting approaches. By Staudinger ligation (A), strain promoted alkyne–azide cycloaddition (B)and inverse electron demand Diels–Alder reactions (C). Covalent bond between the tumour bound pretargeted mAb conjugates and the radiolabelledeffectors are formed at the tumour site.


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on the pretargeting strategy using bioorthogonal reactions. Addi-

tionally, we cover the very recent developments on bioorthogonal

tumour pretargeting that were not included in the previous review

articles.

4.1 Staudinger ligation

In 1919, Staudinger and Meyer reported for the first time a

reaction between an azide and a triphenylphosphine resulting

in an iminophosphorane intermediate which undergoes hydro-

lysis spontaneously in the presence of water to give a primary

amine and corresponding phosphine oxide. More than 80 years

later, Bertozzi and co-workers were the first to realise the potential

of this reaction for biological applications.43 The classical

Staudinger reaction satisfy many of the above mentioned criteria:

it occurs rapidly in water at room temperature and both reactants

are abiotic and react selectively without cross-reaction with bio-

logical media or cell surface proteins. Therefore, this reaction

holds the promise for being applicable in biological environ-

ments. However, the main limitation of this reaction was the

aqueous instability of the iminophosphorane covalent adduct,

that hydrolyses in the presence of water. Using an engineered

triarylphosphine consisting of an ester group as electrophilic

trap within the phosphine, Bertozzi and colleagues were able to

trap the iminophosphorane intermediate intramolecularly, so

that it results in a stable covalent adduct after the rearrangement.

Incorporation of this specific modification transformed the

Staudinger reaction into the Staudinger ligation which is

sometimes also termed as the Staudinger–Bertozzi ligation

(Fig. 9A).43 Over the years, this click reaction became a very

popular tool and found a widespread use including metabolic

labelling of cell surfaces in vitro and in vivo.43

In 2011, van Dongen and co-workers were the first to make

an attempt to evaluate the possibility of the application of

the Staudinger ligation for tumour pretargeting. The authors

therefore modified anti-CD44v6 mAbs with several triazide

functionalities and developed a series of rapidly clearing radio-

labelled phosphines as effector molecules. In order to evaluate

the ligation efficiency in vivo, radiolabelled phosphines were

injected in non-tumour bearing mice 2 h after the administra-

tion of triazide–mAbs. Unfortunately, no evidence of in vivo

Staudinger ligation was detected. These negative results pre-

sumably originate from low reaction rates of the Staudinger

ligation at low reagent concentrations. Moreover, rapid oxida-

tion of phosphine to unreactive phosphine oxide in the blood

was also observed representing another concern of this ligation

strategy.45

4.2 Strain promoted alkyne–azide cycloaddition (SPAAC)

A click reaction between cyclooctyne and azide to form a 1,2,3-

triazole unit was first reported by Krebs and Wittig in 1961

(Fig. 9B). The reaction is analogues to the original copper

mediated Huisgen [3+2] alkyne–azide cycloaddition. However,

it proceeds without the need of Cu, which is toxic to living

tissues, but via the release of the strain of the cyclooctyne

moiety. The reaction is termed as strain promoted alkyne–azide

cycloaddition (SPAAC). Furthermore, the reagents are more

compatible to in vivo conditions and the rate of the reaction is

superior compared to the Staudinger ligation.43 At the moment, to

the best of our knowledge, there are only two reports where SPAAC

was evaluated for tumour pretargeting and the findings were

described in recent reviews.42,45

In 2013, the group of Rossin and Robillard prepared triazide-

conjugated anti-CD20 mAbs (Rituximab) as well as a number of177Lu-labelled DOTA-conjugated cyclooctyne derivatives to assess

the reactivity profile of the triazide–mAbs and the radiolabelled

effector probes in the circulation in non-tumour-bearing mice.

However, the low in vivo reactivity of the generated cyclooctyne

effectors towards azides and their covalent and non-covalent

interactions with serum proteins impeded a sufficient bond

formation between the azido-conjugated mAbs and the 177Lu-

probes in this study.42,45 Soon after that, a report by Kim and

co-workers demonstrated that SPAAC indeed holds promises

for application in tumour pretargeting. In contrast to the study

of Rossin and Robillard, the authors did not use mAb con-

jugates to pretarget the tumour but mesoporous silica nano-

particles that passively accumulate and retain in tumours

through the enhanced permeability and retention (EPR) effect.

These nanoparticles were PEGylated as well as functionalised

with dibenzocyclooctyne (DBCO) and subsequently intravenously

administered into tumour-bearing mice. After a pretargeting

interval of 24 h, the radiolabelled effectors, 18F-labelled azides,

were injected. Following the postinjection interval, PET-CT imaging

indicated an accumulation of radioactivity at the tumour side in

mice pretargeted with the nanoparticles, whereas much less

radioactivity was deposited in the tumours of mice without

nanoparticle pretreatment.42,45 This later study provides a good

indication that SPAAC could, indeed, be used for tumour

pretargeting provided an appropriate cyclooctyne moiety with

favourable reaction kinetics is chosen and a suitable protocol is

applied.

4.3 Inverse electron demand Diels–Alder reaction (IEDDA)

In 2008, the bioorthogonality of inverse electron demand Diels–

Alder reaction (IEDDA) between trans-cyclooctene (TCO) and

tetrazine (Tz) forming pyridazines (Fig. 9C) was disclosed for

the first time.43 The reaction proceeds with a fast rate without

the need of a catalyst, in water, cell media, or in cell lysate and

was found to have a high functional group tolerance. In fact,

IEDDA was shown to be the most promising among the three

bioorthogonal reactions investigated in tumour pretargeting,

especially in vivo, to date. Despite promising results of IEDDA-

mediated pretargeting in cultured cells, its successful transla-

tion into animal requires the IEDDA to satisfy more demanding

criteria: (i) very fast reaction kinetics at low reagent concentra-

tions, (ii) high stability of TCO and Tz moiety in vivo, (iii) prolong

circulation of TCO tagged mAbs, (iv) faster accessibility to

the tumour tissue and (v) rapid clearance of the circulating

Tz-radiolabelled effector molecule from the body.

The in vivo application of IEDDA for pretargeted radio-

diagnostic imaging was first demonstrated in a landmark study

by Robillard and co-workers in 2010. Therein the authors

described the preparation of TCO-conjugated mAbs (CC49)


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directed against the pan-adenoma TAG-72 antigen as target

specific component. 111In-labelled DOTA-Tz probes represent

the small, fast clearing effector component of this pretargeting

approach. The CC49–TCO conjugates were injected into mice

bearing colon cancer xenografts and a pretargeting interval of

1 day was set to allow for their localisation to the malignant

tissue. Upon injection of the radiolabelled Tz derivative, the two

complementary bioorthogonal reactants rapidly reacted in vivo

and SPECT/CT imaging of live mice 3 h postinjection revealed

a pronounced localisation of radioactivity in the tumour and

in the urinary bladder with limited uptake in blood and non-

targeted tissues, such as the liver.45

Over the last few years, significant modifications have been

introduced to this strategy in order to achieve improved T/NT

ratio. For example, TCO and Tz components have been modified

to enhance the rate of ligation, a chaser (clearing reagents)

has been introduced before administration of the radiolabelled

effectors to clear the circulating antibody–TCO conjugates from

the blood, etc. For details on these advancements up to the year

2014, we direct the readers to consult the excellent review articles

published by Knight and Cornelissen as well as Rossin and

Robillard.42,45

Since the last review article in this field, there have been a

few more studies published on this rapidly growing topic. The

group of Jason S. Lewis at the Memorial Sloan Kettering Cancer

Center in New York developed a pretargeted PET imaging

strategy based on IEDDA. Members of this laboratory have

recently developed two novel 64Cu-labelled Tz ligands, 64Cu-

Tz-PEG7-NOTA and 64Cu-Tz-SarAr. Among these two, the later

possesses excellent pharmacokinetic properties with rapid

renal clearance. In combination with a TCO-modified conjugate

of the colorectal cancer-targeting huA33 mAb, 64Cu-Tz-SarAr was

shown to efficiently delineate malignant tissues in murine xeno-

grafts with a high tumour to background contrast in pretargeted

PET imaging and biodistribution experiments. Importantly, the

dosimetry calculations performed in this study suggested that the

total effective radiation dose is significantly lower compared to

doses deposited by imaging using directly radiolabelled mAbs.46

The same group reported recently on the development of a

novel 18F-based pretargeted PET imaging system. Therein the

authors described the production of immunoconjugates

composed of TCO moieties and mAbs (5B1) specific for the

carbohydrate antigen 19.9 (CA19.9), a biomarker for pancreatic

ductal adenocarcinoma. As complementary bioorthogonal species

to 5B1-TCO, aluminium-[18F]fluoride-NOTA-labelled Tz ligands

were synthesised and injected into CA19.9-expressing murine

xenografts 72 h after the administration of 5B1-TCO. Tumoural

uptake of the radiolabelled effector was immediately evident

already 1 h postinjection and tumour-to-background activity

concentration ratios improved over time.47

Recently, different nanoparticles have been proposed to replace

tumour antigen specific mAbs as target specific component to

target a broad range of different tumour entities.48 Such nano-

particulate materials with in vivo hydrodynamic diameters

above the glomerular filtration threshold and prolonged blood

circulation passively accumulate in tumours through the EPR

effect.49 This process depends on their leaky vasculature and

a poorly functional lymph system and results in increased

permeability to nanomaterials.

A new IEDDA-based pretargeting approach using nano-

particles was very recently introduced by Lin and co-workers.

Instead of using TCO-modified immunoconjugates, the TCO

reactive moiety was encapsulated into supramolecular nano-

particles with a uniform size of B100 nm. Small and fast

clearing effector possessing the complementary bioorthogonal

motif Tz-DOTA was synthesised and radiolabelled with 64Cu.

Their in vivo pretargeting experiment started with intrave-

nous injection of the TCO-functionalised nanoparticles into

xenografted mice. After a pretargeting interval of 24 h, radio-

labelled Tz-DOTA was administered and various PET images

were taken at different time points (Fig. 10). Strong PET signals

were observed at the tumour site with highest tumour to liver

signal ratio after 24 h postinjection. Notwithstanding this

impressive performance, also radioactivity levels in the liver

were observed, most likely due to trapping of the nanoparticles

by the mononuclear phagocyte system (MPS) or transchelation

of 64Cu from DOTA in vivo.48 Although the EPR effect is a

common phenomenon in rodent models using subcutaneous

Fig. 10 (a) Timeline of the injection protocol employed for the pretargetedstudy. (b) MicroPET images of the pretargeted study at various time points.(c) Ratio of PET tumour signal to liver signal of xenografted mice from thepreliminary pretargeted study (n = 4). Adapted with permission from ref. 48copyright American Chemical Society.


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and orthotopic xenografts, its occurrence in human primary

and metastatic lesions may be less widespread and less pro-

nounced. As nanomaterials bypassing the renal filtration process

will, however, inevitably end up in the organs of the MPS (spleen

and liver),49 also these organs will be exposed to radiation upon

injection of the radiolabelled effector molecules as also observed

by Lin and co-workers.48 This crucial aspect has to be kept in

mind especially for therapeutic applications of this pretargeting

system.

In addition to the detection of tumour-bound fluorescent or

radioactive effector molecules, the pretargeting concept has

recently been extended to ultrasound imaging. In order to

demonstrate the capture of gas-filled micron-sized bubbles by

means of IEDDA, the group of John F. Valliant developed

Tz-functionalised microbubbles and modified vascular endo-

thelial growth factor receptor 2 (VEGFR2) specific mAbs with

TCO. In their in vivo study, VEGFR2-positive murine xenografts

were pretargeted using the TCO-conjugated mAbs and 24 h

later the Tz-functionalised microbubbles were injected intrave-

nously. Ultrasound images acquired 4 min thereafter showed

high retention of the microbubbles in vascularised regions of

the tumours with significant contrast enhancement compared

to mice without TCO–mAb pretreatment.50

5. Conclusion and outlook

Preclinical and clinical studies have clearly indicated that

tumour pretargeting can overcome important limitations of

conventional directly radiolabelled mAbs. Since its introduction

in the mid-1980s, various pretargeting strategies have been

developed and progressed steadily over the years.

The system based on bs mAbs and the avidin/streptavidin–

biotin components represents without any doubt the most

advanced tumour pretargeting approach for diagnosis as well

as for therapy in the clinical settings, to date. However, this

system suffers from several drawbacks including induction of

immunogenicity and, therefore, future research should be

directed to tackle this important issue. Emerging pretargeting

systems based on hybridisation of synthetic complementary

oligonucleotides or bioorthogonal chemistry in vivo have the

potential to circumvent many of the limitations associated with

the avidin/streptavidin–biotin system. These approaches are

still in the preclinical stage, but possess immense potential.

Regarding bioorthogonal chemistry, IDEEA appears to be the

most suitable of all reactions tested so far for pretargeting due

to easy synthetic access of the required substrates, in vivo

stability of the TCO and Tz moieties and relatively fast reaction

kinetics. However, the kinetics of IDEEA still need further

improvement to become truly applicable in the clinic. This

could be achieved, for example, by introducing suitable modi-

fications either on the TCO or on the Tz moieties. Since the

pretargeting approach is a rapidly growing field, new bioortho-

gonal reactions with even faster reaction kinetics and better

in vivo compatibility will certainly be discovered in the near

future.

As the special strength of tumour pretargeting is the sub-

stantial reduction of healthy tissue exposure, it is in particular

useful for therapeutic purposes. At this point, it is worth

emphasizing that conventional treatments such as chemo-

therapy, external beam radiation and surgery still play a key

role in cancer management. As tumour pretargeting is suitable

for detecting tumours and metastases of minimal or micro-

scopic size, it can be indeed applied to treat and eradicate

residual disease and metastatic lesions. Furthermore, the pre-

targeting strategy can have a significant impact on the treatment

of malignant tissues that are difficult to remove by surgery

or surrounded by sensitive tissues susceptible to damage. For

therapeutic purposes, it is however of utmost importance to

deposit as much radiation as possible precisely in the tumour

without harming bone marrow, liver, spleen and kidney. Since

the blood clearance of the radionuclide-carrying small effector

molecules occurs sufficiently rapid, bone marrow exposure and

haematological toxicity is less a decisive factor. And as long as

the renal clearable radiolabelled effectors are not retained in the

kidneys or bladder but eliminated in the urine, renal doses are

acceptable.10 However, not only the pharmacokinetic behaviour

of the radiolabelled effector molecules determines the effective-

ness of the pretargeting strategy, but also the in vivo stability of

the radiometal–chelate complexes. For that reason, considerable

efforts should be devoted to the development of very stable

radiometal complexes for conjugation to the fast clearing effec-

tor molecules that permit simultaneously imaging, dosimetry

and treatment of tumours.

Although initial clinical studies of pretargeted RIT have been

very encouraging, its full potentials are yet to be proven using large

clinical trials in the near future, if thereby administered doses are

sufficient to result in a significant improvement in responses. It

should be noted that pretargeting is a relatively complex procedure

that might need exhaustive optimisation for its successful transla-

tion into clinic. The combination of pretargeted RIT with other

treatment modalities such as conventional external beam radia-

tion or chemotherapy might be reasonable. In particular, the

treatment with radiosensitising or anti-angiogenic agents can

produce synergistic effects when combined with pretargeted RIT.

With regard to oncotropic vector molecules, tumour antigen

specific mAbs represent an essential platform to precisely detect

and treat tumour malignancies due to their excellent target

specificity and affinity.4 To preserve this unmatched perfor-

mance, their chemical modification necessary to create the

desired mAb conjugates must not affect their immunoreactivity

and affinity and should result in a homogenous population

of bioconjugates. However, conventional bioconjugation such

as random coupling to lysine or cysteine residues result in

complex mixtures of mAb conjugates with possibly divergent

pharmacokinetic behaviour, stability and immunoreactivity.

In contrast, controlled, site-specific bioconjugation strategies

including chemoenzymatic methods have the potential to yield

more homogeneous products and should be pursued conse-

quently for the production of next-generation mAb conjugates.

Another serious problem of the pretargeting approach, which

will need to be dealt with in the future, is the partial cellular


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internalisation of mAbs upon specific interaction with their

respective cell surface-located receptors. Antigen–mAb pairs

known to internalise readily to a large extent are hence sub-

optimal for pretargeting as the prelocalised mAb conjugates

must remain accessible to form the radioimmunoconjugates

with the radiolabelled effector molecules.11 In consequence,

a whatsoever modification of mAb conjugates preventing

their receptor-mediated internalisation would represent a

key milestone for tumour pretargeting.

Finally, pretargeting approaches based on mAbs are limited

to detect and treat populations of cancerous cells with unique

or highly abundant surface markers that are located on the cell

surface of at least some, ideally on the majority of tumour cells

and, most importantly, that are readily accessible by circulating

agents. A substantial advantage of delivering therapeutic radio-

nuclides, especially b-particles, is their long range in tissues

(up to 12 mm). This allows for the irradiation of tumour cells

including cancer stem cells located adjacent to cells to which

the radioimmunoconjugates are attached. Consequently, also

tumour cell subtypes lacking the addressed antigens are

exposed to radiation.6 Targeting of a single type of cell surface

receptors may, however, allow some cancer cells to survive by

downregulation or mutation of the addressed antigen and may

permit renewed tumour growth and clinical relapse. Pretargeted

radioimmunotherapy should thus be applied in combination with

other anticancer therapies and should ideally hit a set of different

tumour-associated antigens.

In summary, there is much space to optimise the main

parameters and improve the effectiveness of the pretargeting

approach. Only if significant benefits for patients can be clearly

illustrated in clinical studies, will this technology be translated

into practise and impact in the clinics.

Acknowledgements

We thank Dr Maik Schubert for providing a substantial part of

the topic’s bibliography. This work was financially supported by

the Swiss National Science Foundation (Professorships No.

PP00P2_133568 and PP00P2_157545 to G. G.), the University

of Zurich (G. G.), the Stiftung fur wissenschaftliche Forschung

of the University of Zurich (G. G.), the Helmholtz Virtual

Institute NanoTracking (Agreement no. VH-VI-421) as well as

the research initiative Technologie und Medizin – Multimodale

Bildgebung zur Aufklarung des in-vivo-Verhaltens von polymeren

Biomaterialien of the Helmholtz-Portfoliothema.

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